Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two α-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2 which are differently sensitive to neonicotinoid insecticides. We suggested that their sensitivity to neonicotinoids could be associated to their intracellular regulation mechanisms. Unfortunately, the intracellular mechanisms leading to the regulation of insect nAChRs are unknown. We demonstrated for the first time that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. We show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine- and clothianidin-induced currents. Indeed, increased cGMP affects the second part of the biphasic current voltage curve, corresponding to the nAChR2 receptors, while maintaining the guanosine triphosphate (GTP) level with GTP-γ-S increased nicotine currents through nAChR2.
We also demonstrated that inhibition of PKG activity with (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca2+) activity because inhibition of Ca2+ concentration with cadmium chloride (CdCl2) abolished KT5823-induced inhibition mediated by nAChR2. These results suggest that nicotine- and clothianidin-induced currents mediated by both α-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cAMP/PKA and cGMP/PKG pathways.